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Departments of Physiology and Medicine; University of California Los Angeles, School of Medicine; Los Angeles, California 90095
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5 pA/pF at –90 mV) in transgenic vs. control myocytes (1.5 pA/pF). The decay rate of caffeine-induced Ca2+i -transient and INa-Ca was 2.5 times faster in transgenic than in control myocytes. 5 mM Ni2+ was equally effective in blocking INa-Ca in control or transgenic myocytes. In 9 out of 26 transgenic myocytes, but none of the controls, Ca2+ influx via the exchanger measured at +80 mV caused a slow rise in [Ca2+]i triggering rapid release of Ca2+ from the SR. SR Ca2+ release triggered by the exchanger at such potentials was accompanied by activation of transient current in the inward direction. In 2 mM Fura-2–dialyzed transgenic myocytes caffeine-triggered Cai-transients failed to activate INa-Ca, even though the kinetics of inactivation of ICa slowed significantly in caffeine-treated myocytes. In 0.1 mM Fura-2–dialyzed transgenic myocytes 100 µM Cd2+ effectively blocked ICa and suppressed Cai-transients at –10 or +50 mV. Our data suggests that in myocytes overexpressing the exchanger, the content of intracellular Ca2+ pools and the signaling of its release by the Ca2+ channel vis-à-vis the Na+-Ca2+ exchanger were not significantly altered despite an up to ninefold increase in the exchanger activity. We conclude that the exchanger remains functionally excluded from the Ca2+ microdomains surrounding the DHP/ryanodine receptor complex.
Key Words: ventricular myocytes • Ca2+ channel • whole cell patch clamp • immunofluorescence • isolated sarcolemmal vesicles
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; Levesque et al., 1994; Lipp and Niggli, 1994; Sham et al., 1992). This controversy may be related, in part, to different levels of expression of the exchanger in different species (Sham et al., 1995b). Ca2+ cross signaling experiments between the DHP and ryanodine receptors in myocytes dialyzed with high concentrations of Ca2+ buffers, however, suggest that the exchanger protein might be excluded from the "functional" microdomain of Ca2+ surrounding the DHP/ryanodine receptor complex (Sham et al., 1995a; Adachi-Akahane et al., 1996), even though immunofluorescence techniques have suggested that the exchanger is located in the t-tubular system near diadic or triadic junctures (Frank et al., 1992; Kieval et al., 1992).
The availability of the cDNA clone for the Na+-Ca2+ exchanger (Nicoll et al., 1990) allows new approaches to physiological problems regarding the exchanger. For example, antisense oligonucleotides have been used to "knock out" exchange activity in both cardiac and arterial myocytes (Lipp et al., 1995; Sidzinski et al., 1995). Transgenic mouse technology offers another opportunity to manipulate Na+-Ca2+ exchange activity. Here we describe the production and characterization of transgenic mice overexpressing Na+-Ca2+ exchange activity specifically in cardiac muscle. Myocytes from the transgenic mice have increased INa-Ca and were used to further probe the ability of INa-Ca and the Ca2+ channel to trigger Ca2+ release. Even with the increased density of INa-Ca, the exchanger failed to trigger Ca2+ release from the sarcoplasmic reticulum (SR)1 in the physiological voltage range.
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) under the control of the 
-myosin heavy chain (-MHC) promoter. The -MHC promoter consisted of 4.5 kb of 5' upstream sequence plus 1 kb of the -MHC gene encompassing exons 1 through 3 of the 5' untranslated region (Gulick et al., 1991; Subramaniam et al., 1991). The presence of upstream introns and exons may improve expression of a transgene (Palmiter et al., 1991). Downstream from the -MHC promoter was the SV40 transcriptional terminator to provide a polyadenylation signal. The exchanger open reading frame was removed from pTB11 (Nicoll et al., 1990) by digestion with EcoRV and SnaBl and ligated into the SalI site between the -MHC promoter and the SV40 transcriptional terminator. The SalI site had first been digested, blunted, and dephosphorylated. Proper orientation of the exchanger insert was confirmed by restriction mapping. The transgene was purified by GeneClean (Bio 101, La Jolla, CA) and microinjected into the nuclei of C57Bl/6xC3HF1 mice by the UCLA Transgenic Core Facility for transgenic mouse production.
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). Thus, on the Southern blot, the 0.4-kb probe would hybridize with the 0.4-kb fragment derived from the EcoRI-digested transgene which contains no exchanger introns. Digestion of the endogenous mouse exchanger gene, however, with EcoRI did not produce a similar sized fragment. Prehybridization and hybridization were carried out as described previously (Li et al., 1994).
Northern Blot Analysis
Total RNA was isolated from mouse tissues by the method of Chomczynski and Sacchi (1987). The RNA was separated on a denaturing 1% agarose gel and transferred onto a Hybond-N nylon filter. The same 0.4-kb cDNA fragment used in the Southern blot analysis above was also used for Northern blot analysis. Hybridization conditions were also the same.
Western Blot Analysis
Proteins were first separated on a 7.5% gel by SDS-PAGE and transferred onto nitrocellulose for 30 min at 100 V. Washings and antibody incubations were carried out in the presence of 1% milk. The primary polyclonal antibody (
) was raised against the canine cardiac exchanger and has been described previously (Philipson et al., 1988). The secondary antibody was goat anti–rabbit IgG coupled to horseradish peroxidase. Diaminobenzidine was used as substrate for color development.
Indirect Immunofluorescent Labeling
Isolated mouse myocytes, either from control hearts or transgenic hearts, were fixed with 2% buffered formaldehyde for 15 min. The fixed cells were quenched in Na+ borohydrate, treated with Triton X-100, and exposed to blocking solution and the monoclonal antibody R3F1 (1/500 dilution) against the Na+-Ca2+ exchanger, as previously described (Frank et al., 1992; Chen et al., 1995). The cells were incubated with fluorescein-labeled goat anti–mouse secondary antibody for 45 min, rinsed, and mounted on glass slides with 90% glycerol plus a photobleaching inhibitor. The confocal fluorescence microscopy was carried out with a Nikon photomicroscope equipped with a molecular dynamic confocal imaging system.
Transport Measurements in Isolated Vesicles
A crude membrane fraction was prepared for measurement of Na+-Ca2+ exchange fluxes. Mouse hearts (100 mg) were homogenized in 1.4 ml of 560 mM NaCl, 10 mM Mops/Tris, pH 7.4, and spun in an Eppendorf centrifuge for 4 min at 11,000 rpm. The pellet was resuspended in 0.9 ml of 140 mM NaCl, 10 mM Mops/Tris, pH 7.4 and spun for 4 min at 11,000 rpm. The pellet was resuspended in 0.8 ml of the same solution and spun briefly (5 s) at 4,000 rpm to remove particulate material. The supernatant, containing Na+-loaded membrane vesicles was used directly for Ca2+ uptake measurements. The protein yield in the final fraction was identical for the control and transgenic mice.
To measure Na+ gradient–dependent 45Ca2+ uptake, 10 µl of the supernatant was rapidly diluted into a Ca2+ uptake medium containing 140 mM KCl, 10 µM 45Ca2+, 1 µM valinomycin, 10 mM Mops/Tris, pH 7.4. The reaction was quenched after 3 s and then filtered. A blank was subtracted in which the uptake medium contained NaCl instead of KCl. We have used this technique extensively in the past to quantitate vesicular Na+-Ca2+ exchange (Li et al., 1991).
Isolation of Adult Mouse Ventricular Myocytes
Adult mouse ventricular myocytes were isolated according to a previously described method (Mitra and Morad, 1985) with minor modification. After injection of heparin sodium (1,000 U/ kg, i.p.), mice were deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.), hearts were quickly excised and perfused in a Langendorff apparatus (1.2–1.6 ml/min) first with nominally Ca2+-free Tyrode's solution composed of (in mM) NaCl, 137; KCl, 5.4; HEPES, 10; MgCl2, 1; glucose, 10; pH 7.3 at 37°C for 7 min, then with Ca2+-free Tyrode's solution containing collagenase (0.5–0.6 Us/ml) and protease (0.55 Us/ml) for 15 min, and finally with low Na+ Tyrode's solution. The ventricular part of the digested heart was then cut into several sections and gently agitated to dissociate cells. The freshly dissociated cells were stored at room temperature in low Na+ Tyrode's (in mM: 52.5 NaCl, 4.8 KCl, 1.19 KH2PO4, 1.2 Mg SO4, 11.1 glucose, 10 HEPES, 145 sucrose, pH 7.4) containing 0.2 mM Ca2+ and were used for up to 10 h after isolation. In all of the electrophysiological experiments, the transgenic mouse line H was used.
Current Recording
Ca2+ current was measured in the whole cell configuration of the patch-clamp technique (Hamill et al., 1981) using a DAGAN 8900 amplifier (Dagan Corp., Minneapolis, MN). The patch electrodes, made of borosilicate glass capillaries, were fire-polished to have a resistance of 1.2 to 2.0 M
when filled with the internal solution composed (in mM): CsCl, 110; tetraethylammonium chloride (TEA-Cl), 30; NaCl, 10; HEPES, 10; MgATP, 5; cAMP, 0.2; K5Fura-2, 0.1–2.0 and titrated to pH 7.4 with CsOH. Inward rectifier K+-current was suppressed by either addition of Ba2+ (0.1 mM) to or omission of K+ from the external solutions. Na+-current was mostly suppressed by addition of 10 µM tetrodotoxin to the external solution, and by including a high concentration (200 µM) of cAMP in the internal solution (Schubert et al., 1989). Myocytes were dialyzed with 200 µM cAMP not only to enhance ICa but also to fully activate Ca-ATPase activity through phosphorylation of phospholamban.
Generation of voltage-clamp protocols and acquisition of data were carried out using pCLAMP software (version 5.5-1; Axon Instruments, Inc., Foster City, CA). The leak currents were not digitally subtracted by the P/N method (N = 5–6) as to avoid suppression of maintained components of INa-Ca. Thus, we chose cells which had little or no leak currents. The series resistance was 1.5 to 3 times the pipette resistance and was electronically compensated through the amplifier. Sampling frequency was 0.5–2.0 kHz, and current signals were filtered at 10 kHz before digitization and storage.
Intracellular Calcium Measurements
Intracellular calcium activity was measured according to the method described earlier (Cleemann and Morad, 1991). Ventricular myocytes were dialyzed with 0.1–2.0 mM Fura-2 via the patch pipettes. Ultraviolet light originated from a 100 W mercury arc lamp, was split into two beams using a mirror vibrating at 1,200 Hz, and passed through the interference filters (410 and 335 nm, 20 nm bandwidth). The fluorescent light passed through a wide-band interference filter (510 nm, 70 nm bandwidth) and was detected with a photo-multiplier. The signal from the photo-multiplier was demultiplexed (Cleemann and Morad, 1991), yielding two signals corresponding to the two wavelengths of excitation. These signals were acquired simultaneously with the whole-cell currents using pCLAMP software. Cai -transients were quantified using FURA 2N program (Adachi-Akahane et al., 1996).
The data collected with dual wavelength excitation of Fura-2 were analyzed to determine the intracellular Ca2+ activity ([Ca2+]i) by the ratiometric method with a Kd value of Fura-2 for Ca2+ as 220 nM (Grynkiewicz et al., 1985). The background fluorescences (F410,bg and F335,bg) were measured after making a giga-seal just before rupture of the membrane. Calibration measurements were performed with samples of 50 µM Fura-2 either saturated with 5 mM Ca2+ (F410,Ca and F335,Ca) or in free form with 10 mM EGTA (F410,EGTA and F335,EGTA).
Drugs were dissolved in the external Tyrode's solution, and applied rapidly using a concentration-clamp device (Cleemann and Morad, 1991).
All the experiments were performed at room temperature (22–25°C).
Collagenase (type A) was purchased from Boehringer-Mannheim Biochemicals (Indianapolis, IN), Protease (type XIV, pronase E) and MgATP from Sigma Chemical Co. (St. Louis, MO), thapsigargin and tetrodotoxin from Calbiochem Corp. (La Jolla, CA), and K 5Fura-2 salt from Molecular Probes, Inc. (Eugene, OR). Thapsigargin was dissolved in DMSO and stored as 10–3 M stock solution. The highest (0.1%) concentration of DMSO used had no effect by itself on ICa or Cai-transients.
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-MHC promoter. This promoter has been used previously in transgenic experiments for cardiac-specific expression (Milano et al., 1994; Soonpaa et al., 1994; Koch et al., 1995). Eight transgenic mouse lines, called lines A through H, were generated. The transgenic mice were all heterozygous so that in all cases nontransgenic littermates could be used for controls. The mice had no readily observable altered phenotype; body and heart weights were all normal. No differences between the transgenic mouse lines were detected unless specifically mentioned below. Table I compares some electrophysiological properties of the myocytes not directly related to the exchanger activity in control and transgenic mice. The size of the myocytes (cell capacitance), the resting Ca2+ concentrations (basal [Ca]i), the magnitude of triggered Cai-transients (
Cai), and the Ca2+ current density (ICa) were not significantly altered in transgenic mice.
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Fig. 2 A shows a Northern blot with RNAs isolated from the hearts of transgenic mice and their nontransgenic littermates from six different lines probed with an exchanger cDNA probe. A strong signal from RNA isolated from the transgenic hearts is seen at 3 kb after an exposure of only 1 h. With such short exposure times, no signal is observed with the RNA from the control mice. A signal from the native exchanger becomes visible at 7 kb in all lanes after longer exposures (not shown, but see Fig. 2 B). Similar results were obtained for transgenic mouse lines B and E. Clearly, substantial amounts of RNA are being transcribed from the transgene.
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-MHC promoter is supposed to permit gene expression only in cardiac tissue, therefore we next assessed the tissue specificity of exchanger transgene expression. Fig. 2 B shows the results of a Northern blot analysis using RNA isolated from the heart, lung, brain, or skeletal muscle of transgenic mice or nontransgenic littermates from line H. In this case, a longer exposure time was used to permit visualization of both the native and transgenic Na+-Ca2+ exchangers. In the tissues from the nontransgenic mice, a weak signal at 7 kb from the native exchanger is seen only in the RNA from the cardiac tissue. (Upon longer exposure, 7-kb bands also become visible in the RNA from brain and lung.) In the RNAs from the transgenic mice, a strong signal is seen only with the cardiac RNA at 3 kb (transgenic exchanger) and a weak signal is seen at 7 kb (native exchanger). Not visible in the photograph, but discernible by eye, is a low level of expression of the 3-kb transgenic exchanger in the lung of transgenic mice. This was also seen in lung RNA from other transgenic mouse lines. Thus, the -MHC promoter was not completely silent in lung tissue. In one transgenic mouse line (line E), expression of transgene transcripts were much higher in lung than in the other transgenic lines though still many fold lower than the expression level in heart. Line E was also the only line in which transgene transcripts could also be weakly detected in brain RNA. Subramaniam et al. (1991) have previously noted a low level of -MHC gene expression in lung tissue, specifically in the thick wall of the pulmonary veins of the lung. Nevertheless, of those tissues tested, high levels of transgenic Na+-Ca2+ exchanger transcript were found only in the myocardium.
Western blot analysis.
Immunoblots were performed to assess the level of Na+-Ca2+ exchanger protein in the hearts of the transgenic mice. The proteins of myocardial homogenate were separated by SDS-PAGE and probed with a polyclonal antibody to the canine sarcolemmal Na+-Ca2+ exchanger. Strong immunoreactivity is seen in the transgenic hearts but not in the hearts from control littermates (Fig. 3). The protein bands which immunoreact have a similar pattern to that seen with isolated canine sarcolemmal membranes, a positive control (Philipson et al., 1988). The transgenic exchanger protein bands, however, appear to be of slightly smaller molecular weight than those of the isolated sarcolemma, perhaps due to a small difference in amount of glycosylation. We have previously demonstrated that glycosylation does not affect exchanger function (Hryshko et al., 1993).
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Immunofluorescence
Overexpression of the Na+-Ca2+ exchanger is also clearly demonstrated by immunofluorescence (Fig. 4). The confocal micrographs of the control (Fig. 4 A) and transgenic (Fig. 4 B) myocytes were taken under identical conditions. In the transgenic myocytes, there is intense labeling of both the surface and t-tubular sarcolemma as well as the area surrounding the nucleus which is presumably the Golgi apparatus involved in protein trafficking. As described above, the antibody reactions cannot be used for quantitative comparison of exchanger expression.
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). This preferential localization is much less obvious in mouse myocytes in which strong staining of the peripheral sarcolemma is also observed. This species difference may be related to the large amount of peripheral sarcoplasmic reticulum found in mouse myocytes ( J.S. Frank, unpublished observations). Thus, even the Na+-Ca2+ exchangers found in surface sarcolemma in mouse myocytes may be in close proximity to underlying sarcoplasmic reticulum.
Evidence for functional overexpression of the Na+-Ca2+ exchanger.
Two types of experimental protocols using freshly isolated ventricular myocytes and one set of experiments using a crude membrane preparation were employed to examine the exchanger activity in control and transgenic mice.
In the first set of experiments we prepared a crude myocardial membrane fraction and assayed for Na+ gradient-dependent 45Ca2+ uptake. In a second set of experiments, Fura-2-dialyzed myocytes were clamped at holding potentials of –90 mV and were subjected to rapid (<50 ms) application of 5 mM caffeine to induce Ca2+ release from the SR to activate a transient Ni2+-sensitive inward INa-Ca (Ca2+-extrusion mode of the exchanger; Callewaert, Cleemann and Morad, 1989). In a third set, long (1–2 s) depolarizing pulses to positive potentials were used to measure the Ca2+ influx mode of the exchanger. The maintained outward Ni2+-sensitive current and the accompanying rise in [Ca2+]i were quantified to represent Ca2+ transported by the exchanger (Kimura et al., 1986; Näbauer and Morad, 1992).
Na+-Ca2+ exchange activity in cardiac membrane fraction.
We prepared crude membrane fractions and assayed Na+ gradient-dependent 45Ca2+ uptake. Results obtained using hearts from transgenic mouse line H are shown in Fig. 5. Na+-Ca2+ exchange activity is 148% higher in vesicles from transgenic hearts than in vesicles from the hearts of control littermates. The level of overexpression in vesicles from transgenic hearts from other mouse lines averaged about 100%, though due to scatter, it was not clear if there were significant differences. The level of overexpression as assessed electrophysiologically from line H mice (see Table II) appears to be somewhat larger than that measured in vitro by isotope flux.
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).
Fig. 7 illustrates ICa-gated Ca2+ release in two myocytes obtained from transgenic and non-transgenic littermates. In both cell types the magnitude of ICa and Cai-transients were similar (see also Table I). In transgenic cells, however, ICa measured between –20 and +20 mV was consistently followed by a slowly activating "transient inward current" during the rise of [Ca2+]i. Further, a slowly decaying tail current was observed on repolarization of the membrane. Both the "transient inward current" and the slowly decaying tail currents were abolished by depletion of SR Ca2+ stores by incubation of myocytes in thapsigargin or caffeine (data not shown). A similar (intracellular Ca2+ store-dependent) transient inward current and large slowly inactivating inward tail currents, abolished by replacement of extracellular Na+ with Li+, were also reported in myopathic hamster myocytes overexpressing the exchanger (Hatem et al., 1994).
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In myocytes from control mice (Fig. 8 A) a small Ni2+-sensitive INa-Ca consistently accompanied a small rise in [Ca2+]i in thapsigargin-treated myocytes (Fig. 8 B). Even though the rise in myoplasmic [Ca2+] induced by the exchanger was small in control myocytes (Fig. 8 A), repolarizing to –80 mV activated ICa "tails" triggering significant release of Ca2+. In thapsigargin-treated myocytes, the rise in [Ca2+]i in response to depolarization was somewhat larger than in control myocytes (Fig. 8 B), but Ca2+ release triggered by Ca2+ channel tail current was absent (compare Fig. 8 A with B). These results suggest that the small rise of [Ca2+]i induced by the influx of Ca2+ via the exchanger in control mice is blunted by the SR activity. In transgenic myocytes, however, depolarizing pulses to less positive voltages (+60 mV, Fig. 8, C and D) produced much larger rises in [Ca2+]i, often triggering Ca2+ release from the SR (Fig. 8 C) which activated a transient current in the inward direction at +60 mV, representing Ca2+ extrusion by the exchanger (Fig. 8 C). 6–8 min exposure of transgenic myocytes to 1.0 µM thapsigargin completely suppressed Ca2+ release and the inwardly directed transient current deflections (Fig. 8 D). Instead, intracellular Ca2+ slowly but continuously increased to values of 500–600 nM during the depolarizing pulses. 5 mM Ni2+ blocked INa-Ca, completely suppressed the rise in intracellular Ca2+, and abolished the slowly decaying exchanger-generated tail currents following the repolarization (Fig. 8 D).
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Fig. 9 shows that Ca2+ influx via the exchanger is significantly blunted by a functional SR even in transgenic mice. In this myocyte, depolarization from –80 to 80 mV caused only slight increase in [Ca2+]i, although repolarization from 80 to –80 mV triggered Ca2+ release via Ca2+-influx through the deactivating L-type Ca2+ channels. Ni2+ at 5 mM concentration blocked both Ca2+ influx transported via the exchanger and the Ca2+ channels. The same myocyte, treated with 5 mM caffeine however, showed significant rise in [Ca2+]i during the pulse to +80 mV (Fig. 9 B). This large rise of [Ca2+]i was completely suppressed by 5 mM Ni2+.
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; Sham et al., 1995a). The experiment was designed to induce Ca2+ release first by caffeine and then by ICa. Thus it was possible to measure almost simultaneously the magnitude of INa-Ca activated by caffeine, or ICa activated by depolarization, and their respective Cai-transients. Although 2 mM Fura-2 completely suppressed the activation of caffeine-induced INa-Ca, it did not suppress cross signaling between DHP and ryanodine receptors (Fig. 10 B). Fig. 10 B also shows that ICa, activated following the depletion of Ca2+ pools by caffeine, was somewhat larger, triggered a small Cai-transient, and inactivated significantly more slowly (Fig. 10 C ). These results were similar to those observed in control mice myocytes and suggest that even though the density of the exchanger current is strongly enhanced in transgenic mice, the exchanger remains more susceptible to cytoplasmic Ca2+-buffering than the Ca2+ channel. Thus, in the presence of 2 mM Fura-2, the Ca2+ channel continues to signal Ca2+ release (Fig. 10 A), and is in turn regulated by Ca2+ released via the ryanodine receptors (Fig. 10 C ). We conclude that increasing the density of INa-Ca by at least threefold does not give the exchanger the type of access to the ryanodine receptor as that of the Ca2+ channel.
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). Thus it appears that the Ca2+ channel, even in myocytes overexpressing the exchanger, remains the primary pathway for gating the ryanodine receptor.
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Consequences of Overexpression of the Exchanger
Since the exchanger is a major pathway for Ca2+ extrusion from the cytosol, it may be expected that its overexpression would reduce the Ca2+ content of the SR. Table II clearly shows that there was no significant decrease in Ca2+ content of SR as assessed from the magnitude of caffeine-induced Ca2+ release ([Cai], column 3). Such a finding is consistent with the absence of significant changes in cardiovascular phenotypic parameters (e.g., heart rate and blood pressure) in transgenic mice overexpressing exchange activity (H. Rockman, personal communication). Table I, also, shows that there were no significant differences in the density of Ca2+ channel current in control vs. the transgenic myocytes. This finding supports the observation of a recent report (Silverman et al., 1995) that the duration of the action potential in these transgenic myocytes does not change significantly at 50% duration, a period where ICa may be the predominant inward current. Prolongation of the action potential measured at 90% of its duration reported in the same study may have been caused by the larger inward exchanger current (see Fig. 7).
Overexpression of the Exchanger and Ca2+ Microdomains of DHP/Ryanodine Receptors
Recent data using high concentrations of Ca2+ buffers suggests that Ca2+ signaling in cardiac muscle occurs via microdomains of Ca2+ (Sham et al., 1995a; Adachi-Akahane et al., 1996). Fig. 10 shows that the release of Ca2+ by rapid application of caffeine in transgenic myocytes failed to activate the inward exchanger current in the presence of 2 mM Fura-2 (e.g. Fig. 6), even though the kinetics of inactivation of ICa were markedly altered after the release of Ca2+ from the SR. One possible interpretation is that the release of Ca2+ from the ryanodine receptor is effectively buffered by 2 mM Fura-2, placing the exchanger at microdomains outside of those surrounding DHP and the ryanodine receptor. The differential sensitivity of the exchanger for Ca2+ transport and the Ca2+ channel to Ca2+-induced inactivation, however, may also contribute to the data of Fig. 10. The sensitivity of Ca2+ sites on the two proteins, however, suggests about 5 µM affinity for the Ca2+ transport site of the exchanger (Matsuoka and Hilgemann, 1992) vs. much higher Ca2+ for Ca2+ channel inactivation (10–15 µM Haack and Rosenberg, 1994; and 50–100 µM Morad et al., 1988). Thus the failure to activate INa-Ca while strongly modulating the kinetics of inactivation of Ca2+ channel is more consistent with the idea that the exchanger remains excluded from the Ca2+ microdomains surrounding the DHP/ryanodine receptor complex, even in the transgenic mice.
Physiological Role of the Exchanger in Transgenic Myocytes
One reason for developing these transgenic mice was to enhance the exchanger activity in the Ca2+-influx mode. The activity of the exchanger was enhanced in most myocytes to levels where the density of current carried by the exchanger in the Ca2+ efflux mode was about 5 pA/pF, compared to 1.6 pA/pF in control myocytes (see Table II). Assuming that a similar increase in the activity of the exchanger takes place at positive potentials (Ca2+ influx mode of the exchanger), densities of current equivalent to those of Ca2+ current may be generated by the exchanger.
Ca2+ influx via the exchanger when activated by large and long depolarizing pulses did trigger Ca2+ release in 9 out of 26 transgenic myocytes. However, 100–300 ms were required for the exchanger to activate the Ca2+-induced Ca2+ release mechanism (Fig. 8). In part, because of relatively low capacity of the exchanger versus that of the SR Ca2+ pump (which may prevent significant accumulation of cytosolic Ca2+), the exchanger fails to trigger Ca2+ release on beat-to-beat basis, especially at high mouse heart rates (6 Hz). In the physiological range of membrane potentials, –10 to +20 mV, we consistently failed to produce sufficient influx of Ca2+ via the exchanger to trigger Ca2+ release in transgenic myocytes dialyzed with 0.1 mM Fura 2 (Fig. 11), even though Ca2+ release triggered by Ca2+ current induced a large inward exchanger current (Fig. 7). Even though the exchanger may not have direct access to Ca2+ microdomains of the DHP/ryanodine receptor complex even when overexpressed, the finding that Ca2+ release in transgenic myocytes was significantly enhanced when the exchanger was blocked by Ni2+ (Table II), places the exchanger within distances close enough to the ryanodine receptor to blunt the Ca2+-induced Ca2+-release process. Thus, the overexpressed exchanger appears to produce functional consequences in the Ca2+ efflux, but not in the Ca2+ influx mode.
Satomi Adachi-Akahane's present address is Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.
1 Abbreviations used in this paper:
-MHC, -myosin heavy chain; SR, sarcoplasmic reticulum. 
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant HL48509 and Laubisch Fund to K. Philipson and HL16152 to M. Morad.
Submitted: 21 October 1996
Accepted: 20 March 1997
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-myosin heavy chain gene promoter in transgenic mice, J Biol Chem, 1991, 266, 24613–24620.
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